Efficient electrical contacting of redox-enzymes with electrodes is a key process in the tailoring of enzyme-electrodes for bioelectric applications such as biosensors. As redox-enzymes usually lack direct electrical communication with electrodes, therefore previously many approaches involving the application of diffusional electron mediators, the tethering of redox-relay groups to the protein, or the immobilization of the enzymes in redox-active polymers have been used to establish electrical communication between the redox-proteins and the electrodes. However, relatively inefficient electrical contacting was achieved in these approaches due to the nonoptimal modification of the enzymes by the redox-tethers, or the lack of appropriate alignment of the enzymes with respect to the electrode. Very efficient electrical coupling can be achieved if the enzyme, its cofactor, and the electron mediator are in proper orientation at the electrode. Recently, efficient electrical communication between redox-proteins and electrodes was achieved by the reconstitution of apo-enzymes on relay-cofactor monolayers associated with electrodes.
Dehydrogenases enzymes essential for cellular metabolism are often used as a biocatalyst for chiral chemicals or for sensing applications due to enzymes activity, thermal stability, ability to function in the presence of molecular oxygen. Secondary 30 alcohol dehydrogenases (2° ADH's) are a class of enzymes, using nicotinamide adenine dinucleotide, N AD+, (EC 1.1.1.1), nicotinamide adenine dinucleotide phosphate, NADP+(EC 1.1.1.2), or both (EC 1.1.1.71) as the cofactor. Many dehydrogenase enzymes require the diffusion of the cofactor into the Rossmann fold of the protein. This process allows electrons to be freely transferred between the redox-center of the protein and the cofactor. However, the difficulties associated with in situ regeneration of the enzyme's cofactor have hindered commercial development of dehydrogenasebased biosensors and biocatalytic reactors. Both the direct electrochemical oxidation 5 and reduction of NAD(P)+are kinetically unfavored, requiring the use of high overpotentials. The potential needed for direct oxidation (approximately IV vs Standard Calomel electrode (SCE)) is subject to interference of ascorbic acid and molecular oxygen. The potential needed for oxidation and reduction of NAD(P)+, can be reduced with the use of electron mediators which transfers electrons between the electrode and 10 the cofactor at more moderate voltages (−0.15 to 0.15 V). Suitable mediators include quinones, ferrocenes, phenylendiimines, phenoxazines, toluidine blue (TBO), phenothiazines, catechols, metal complexes, and organic conducting salts. However, there are some fundamental problems with the use of a diffusional electron mediators for electrochemical detection. Many electron mediators such as Meldola's Blue (MB) and 15 toluidine blue (TBO) are known to electropolymerize on the electrode. To overcome this problem electron mediators have been electrochemically tethered to the protein, the electrode, or immobilized in a polymer matrix.
Electrodes have previously been coated with a thin layer of conductive polymers (polypyrrole (PPy) and polyanaline (PA)); these electrodes have been shown to accelerate the oxidation of NADH. Poly(thionine), poly(3,4-di-hydroxybenzaldehyde), poly(metallophthalocyanine), poly(o-aminiophnol)(PAP) and poly(ophenylenediamine) have shown the ability to mediate electron transfer and have been reported to easily form polymer matrices. Polypyrrole (PPy) and PA, along with other conductive polymers which have shown to transfer electrons, are known to change morphology. Other approaches include the incorporation of the electron mediators or cofactors into the polymer matrixes either by physical encapsulation or by covalent modification. Polyelectrolytes, e.g. polyacrylic acid (PAA) and poly(allylamine) hydrochloride (PAH), can be assembled on the surface, while the surface morphology of the polyelectrolyte on the electrode by manipulating the degree of protonation. PAH and PAA can be adsorbed onto any negatively or positively, respectively, charged electrode. The reactive end groups of the polyelectrolytes allow for the fabrication of an electron transfer scaffold.
Several approaches have been developed to facilitate electron transfer between the electrode and enzyme, including the use of a diffusional mediators to shuttle electrons between the electrode and cofactor, immobilizing the enzymes in conductive polymers, and constructing redox relays by attaching enzymatic cofactors inside imprinted polymers. Many enzyme-immobilization methods result in the random orientation of the redox centers of the proteins relative to the electrode. Ideally, interfaces should maintain the mediator, the cofactor, and the enzyme in a proper orientation, prevent degradation and diffusional loss of components, be customizable to adapt to different mediators, cofactors, and enzymes, as well as be inexpensive to fabricate. Zayats et al. assembled on the electrode a linear molecular chain consisting of the mediator, the cofactor and the enzymes, maintaining each of the components in the proper spatial orientation. This approach has been shown to work with flavoenzymes, hemoproteins, as well as pyrrolquinoline quinine (PQQ) containing enzymes. The cofactor, NAD(P)+, was bound to the electrode through a phenylboronic acid affinity linkage with cis-diol functionality of the cofactor. The use of a boronic acid affinity linkage allows the enzyme to bind to the cofactor, allowing efficient, multistep electron transfer, and prevented component losses due to diffusion. However, this approach has the disadvantage of requiring two linkages to be formed with the electron mediator: one with the electrode, and the other with the cofactor.